RNA foci are a result of expanding RNA repeats. The expanded RNA repeats are retained in the nucleus, and adopt unusual secondary structures, sequester various RNA binding proteins, and can become toxic to the cell. RNA-RNA binding protein complexes or aggregates form insoluble nuclear foci which are the cause of cellular defects. The abnormal expansion of nucleotide repeats can lead to numerous effects on genes such as the inhibition of transcription and the loss-of-function of proteins, leading to disease.
More than 30 incurable diseases of the neurological and neuromuscular system are known in humans. Simple microsatellite expansions usually consisting of 3 to 6 nucleotides cause these diseases. The repeats can occur in non-coding regions. The result is a dominantly inherited disease characteristic of a toxic RNA gain-of-function disease.
In humans, the expansion of short tandem repeats of tri-, tetra- and pentanucleotides in single genes cause these hereditary neurological diseases. Numerous central nervous tissues have been found to contain RNA foci. Repeat RNA foci can differ in size, shape, cellular abundance and protein composition but their formation has a negative impact on cellular functions. The expanded repeat tract can also be located in protein-coding sequences and affect the final gene product of the mutant gene.
Walsh MJ, Cooper-Knock J, Dodd JE, et al. Invited Review: Decoding the pathophysiological mechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art. Neuropathology and Applied Neurobiology. 2015;41(2):109-134. doi:10.1111/nan.12187. https://www.ncbi.nlm.nih.gov/pubmed/25319671
Rohilla KJ, Gagnon KT. RNA biology of disease-associated microsatellite repeat expansions. Acta Neuropathologica Communications. 2017;5:63. doi:10.1186/s40478-017-0468-y. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574247/).
Wojciechowska M, Krzyzosiak WJ. Cellular toxicity of expanded RNA repeats: focus on RNA foci. Human Molecular Genetics. 2011;20(19):3811-3821. doi:10.1093/hmg/ddr299. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3168290/.
Fluorescence in situ hybridization (FISH) allows for the detection and localization of specific sequences of nucleic acids inside cells or tissue. FISH antisense probes hybridizing to specific RNA transcript sequences allow distinguishing the presence of particular transcripts the contain simple repeat expansions. FISH enables the detection of a variable number of RNA foci scattered throughout the nucleus in cells expressing mutant RNA. In recent years ribonucleoprotein foci of repeated CUG, CCUG, CGG, CAG, AUUCU and UGGAA motifs present in different tissues have been characterized in more detail.
In myotonic dystrophy type 1 (DM1) which is a multisystemic disease, the cause is an expanded CTG repeat in the 3-untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. The expanded CUG repeat RNA (CUGn RNA) is retained in the nucleus where it forms RNA foci. DM1 leads to defects in regulated alternative splicing events during development. In DM1 the RNA foci sequester the muscleblind like splicing regulator ( MBNL) family of splicing factors and induce upregulation of CELF1 activity through PKC-mediated phosphorylation and altered microRNA regulation.
DM1 and DM2 repeating RNAs are not translated into protein. DM1 is caused by an expansion of a CUG repeat in the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. DM2 is caused by an expansion of a CCUG repeat in intron 1 of the zinc finger 9 protein (ZNF9) pre-mRNA. The model for how these RNAs contribute to DM1 and DM2 describes an RNA gain-of-function that occurs upon expansion. In the model, long, toxic repeats fold into hairpins and bind the RNA splicing regulator muscleblind-like protein 1 (MBNL1). Sequestering of MBNL1 caused by the repeating RNAs causes splicing defects in a subset of pre-mRNAs, including the insulin receptor and the muscle main chloride ion channel.
Figure 1: Crystal Structure of the Triplet Repeat in Myotonic Dystrophy. Synthetic RNA was used for the folding and assembly of the RNA duplex. A sitting drop vapor diffusion method was used for crystallization, and a beam line at the Advanced Photo source at Argonne National Laboratory was used for collecting the diffraction data sets of crystals (Ref.: Myotonic dystrophy type 1 RNA crystal structures reveal heterogeneous 1 x 1 nucleotide UU internal loop conformations. Kumar A, Park H, Fang P, Parkesh R, Guo M, Nettles KW, Disney MD; Biochemistry (2011) 50 p.9928-9935. https://www.ncbi.nlm.nih.gov/Structure/pdb/3SZX).
The MBNL 1 gene encodes a member of the muscleblind protein family which was initially described in Drosophila melanogaster. The encoded protein is a C3H-type zinc finger protein that modulates alternative splicing of pre-mRNAs. Muscleblind proteins bind specifically to expanded dsCUG RNA but not to normal size CUG repeats and may thereby play a role in the pathophysiology of myotonic dystrophy.
MBNL1 gene: https://www.ncbi.nlm.nih.gov/gene/4154
Muscleblind-like protein 1: https://pharos.nih.gov/idg/targets/Q9NR56
Members of this protein family regulate pre-mRNA alternative splicing and may also be involved in mRNA editing, and translation. It is thought that this gene may play a role in myotonic dystrophy type 1 (DM1) via interactions with the dystrophia myotonica-protein kinase (DMPK) gene. Alternative splicing results in multiple transcript variants encoding different isoforms. The members of the CELF/BRUNOL protein family contain two N-terminal RNA recognition motif (RRM) domains, one C-terminal RRM domain, and a divergent segment of 160-230 aa between the second and third RRM domains.
CELF1 gene: https://www.ncbi.nlm.nih.gov/gene/10658
CAG expansion disorders
In the CAG expansion disorders Huntington’s disease (HD), X-linked spinal and bulbar muscular atrophy (SBMA), X-linked spinal and bulbar muscular atrophy (DRPLA), and spinocerebellar ataxia type disorders (SCA1, SCA2, SAC3/MJD, SCA7 and SCA17) the molecular pathogenesis appears to be primarily mediated by a deleterious gain of function of the polyglutamine tract encoded by the expanded trinucleotide sequence.
Triplet Repeat Diseases: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3913379/
Huntington disease: https://ghr.nlm.nih.gov/condition/huntington-disease#genes